Hemophilia A and B, caused respectively by decreased levels of clotting factor VIII (F.VIII) and IX (F.IX) levels in peripheral blood, are the most common severe inherited bleeding disorders. Although purified clotting factors from human plasma can be infused into these patients to prevent or treat bleeding episodes, this poses the risk of spreading of diseases such as Creutzfled-Jakob disease, HIV, and hepatitis C. Although recombinant clotting factor preparations are available, the supply is insufficient to cover the world-wide demand.
As an alternative to generating recombinant clotting factors in cell culture, the use of transgenic animals as bioreactors for the production of clinically useful quantities of proteins has been proposed. The transgenic animal expresses the desired protein in a body fluid, such as milk, from which the protein can readily be isolated. Transgenic pigs that secrete F.VIII (U.S. Pat. No. 5,880,327 to Lubon et al.) or F.IX (Van Cott et al. 1999. Genet. Anal. 15:155-60) in their milk were generated using gene constructs that have regulatory sequences from the gene for mouse whey acidic protein (the 4.2 kb 5′ promoter of WAP), in combination with the cDNA sequence encoding human F.VIII or F.IX. F.VIII pigs secrete 2.7 μg/ml of human F.VIII into milk (Paleyanda et al. 1997. Nat. Biotech. 15:971), while F.IX pigs secrete 2.5 g/L of human F.IX into milk, and the factor is biologically active as measured by an activated partial thromboplastin time assay (APTT).
Therefore, large amounts of functional protein can be obtained from F.IX pigs, which is purified from the milk, and subsequently administered intravenously (i.v.) to a hemophiliac patient. However, it has previously been thought that oral administration of F.VIII or F.IX results in degradation and/or inactivation of the protein due to the acidic and enzymatic composition of the stomach. For example, U.S. Pat. No. 4,348,384 to Horikoshi et al. teaches the administration of an oral composition containing purified F.VIII or F.IX encapsulated in a liposome along with a protease inhibitor, for the purpose of evading gastric inactivation of the therapeutic protein.
One complication that results from i.v. administration of F.VIII or F.IX is that up to 25% of hemophiliacs develop inhibitors, such as antibodies against F.VIII or F.IX that inactivate their procoagulant activity (Fields et al. 2000. Mol. Ther. 3:225-35; Bristol et al. 2001. Hum. Gene Ther. 12:1651-61; Ge et al. 2001. Blood 97:3733-7; Brinkhous et al. 1996. Blood 88:2603-10). Most inhibitory antibodies develop in severely affected patients who have little or no circulating F.IX or F.VIII antigen due to genetic deletion. However, patients with a family history of inhibitor development, severe disease, older age or higher numbers of clotting factor replacement episodes, also have a higher incidence of developing these inhibitors (Roberts. 1997. Inhibitors and their management. In Hemophilia & other inherited bleeding disorders. Rizza & Lowe, eds. W B Saunders Company Ltd., London, p. 365.). The inhibitors can completely inhibit the activity of infused clotting factor and make further treatment difficult.
One means of reducing clotting factor antibodies is the induction of immune tolerance to the clotting factors. For example, Roberts (JAMA 259:84-5, 1988) reviews studies in which F.VIII was injected at high concentrations. Although some studies reported that the level of anti-factor VIII antibodies decreased, due to the high doses required, the method was never widely used because it was prohibitively expensive. Oral administration of F.VIII as a means to prevent the formation of anti-F.VIII antibodies has also been attempted, with mixed results. Oral administration of purified F.VIII to newborn mice did not suppress induction of anti-F.VIII antibodies (Kaplan et al. 2000. Semin. Thromb. Hemost. 26:173-8). Oral administration of purified F.VIII to one of three patients with acquired hemophilia did reduce the amount of anti-F.VIII antibodies (Lindgren et al. 2000. Thromb. Haemost. 83:632-3). However, there are no teachings that oral administration of clotting factors alone can be used to treat hemophilia which is caused by inadequate expression of a clotting factor.
Attempts to induce stable production of the missing clotting factors by gene therapy using injections of transfected myoblasts have also been hampered by the host immune system which, not being tolerant of the missing factor, generates a strong rejection response when introduced (Fields et al. 2000. Mol. Ther. 3:225-35). Thus, inhibitory antibodies to the factors and vigorous T cell responses to the genetically transfected cells are major hurdles to successful treatment of hemophilia.
Another disadvantage to introduction of a foreign gene through the use of a viral vector, is the possibility of an elicited immune response against the vector and/or the transgene product For example, intramuscular (i.m.) injection of an adenoviral vector expressing human F.IX into the hind limbs of hemophiliac mice results in a CTL response against F.IX, and destruction of the transduced cells, whereas i.m. injection of a less immunogenic vector (adeno-associated virus, AAV) expressing human F.IX results in long-term persistence of the transduced cells and an absence of CTL to F.IX (Roberts. 1997. Inhibitors and their management. In Hemophilia & other inherited bleeding disorders. Rizza & Lowe, eds., W B Saunders Company Ltd., London, p. 365.). However, in both cases formation of antibodies neutralizes F.IX activity. The same problem exists in the canine model of hemophilia B, where anti-F.IX antibodies neutralize F.IX activity after gene therapy with F.IX (Evans et al. 1989. Proc. Natl. Acad. Sci. U S A. 86:10095-9; Mauser et al. 1996. Blood. 88:3451-5; Herzog et al. 1999. Nat. Med. 5:56-63; and Kay et al. 2000. Nat. Genet. 24:257-261).